Isolation of cyclase, epimerase and a ring expansion enzyme for producing unnatural cephalosporins

ABSTRACT

The enzymes, cyclase, epimerase and a ring expansion enzyme, are isolated separately from a cell-free extract of a prokaryotic beta-lactam producing organism. The isolated enzymes are used to produce unnatural cephalosporins from polypeptide precursors. Isolation is carried out by adding ammonium sulfate to 40% saturation to the cell-free extract to precipitate contaminating proteins, adding ammonium sulfate to 70% saturation to the resultant supernatant to precipitate the enzymes, suspending the precipitated enzymes in a buffer, separating epimerase from the suspension by gel filtration, and separating cyclase and the ring expansion enzyme from each other by ion exchange chromatography.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a cell-free process for producingcephalosporin antibiotics from polypeptides and derivatives thereof.

2. The Prior Art

The beta-lactam family of natural products includes the penicillins:##STR1## cephalosporins: ##STR2## and cephamycins ##STR3## in which thebeta-lactam ring is fused to a five or six membered sulfur containingring; together with clavulanic acid ##STR4## in which the beta-lactam isfused to a five membered oxygen containing ring;

the carbapenems ##STR5## in which the beta-lactam is fused to a fivemembered carbon containing ring;

and the nocardins and monobactams ##STR6## which are monocycliccompounds. Although there are many members of this family, only two canbe used directly in medicine without structural change. These arepenicillin G, the penicillin in which R=benzyl, and clavulanic acid. Allother clinically important beta-lactam compounds have been prepared fromone or other of the natural products by structural change. For manyyears the changes have been generally effected by substitution aroundthe peripheries of the various ring systems and not in the ring systemsthemselves. Since 1974, however, efforts have been concentrated onnuclear modification of the beta-lactam natural product to producecephalosporins, oxacephems and their derivatives. Such efforts havegenerally resulted in complex chemical processes containing upwards of16 steps with the result that the products are obtained in generally lowyield and at extremely high cost. Moxalactam®, for example, a thirdgeneration cephalosporin, is approximately five times more expensivethan cephalothin, a first generation cephalosporin; and cephalothin is,in turn, approximately fifty times more expensive than ampicillin, asemi-synthetic penicillin (Drug Topics Red Book 1981).

Attention has therefore turned to alternative methods of synthesis, andin particular to microbiological methods. Cell-free syntheses ofpenicillins and the related cephalosporins are known in the art andattention is directed to U.S. Pat. No. 4,178,210 issued Dec. 11, 1979 toA. L. Demain et al, which teaches conversion only of the D-form,penicillin N, to a cephalosporin compound. In U.S. Pat. No. 4,248,966issued Feb. 3, 1981, A. L. Demain et al teach the production ofisopenicillin derivatives, in a cell-free system using an extract fromCephalosporium acremonium, from a tripeptide composed of unsubstitutedor β substituted D-valine, unsubstituted or substituted L-cysteine, andL-α-aminoadipic acid or its analogs. Freezing of the cell-free extractresulted in inactivation of certain enzymes so that conversion did notproceed past the isopenicillin stage. In U.S. Pat. No. 4,307,192 issuedDec. 22, 1981, A. L. Demain et al teach the use of a fresh (i.e. notfrozen) cell-free extract of C. acremonium so as to preserve theracemase (epimerase) agent or agents necessary for the converstion ofisopenicillin N to penicillin N, a necessary intermediate step in theprocess for conversion of L-aminoadipyl-L-cysteinyl-D-valine(abbreviated to LLD in the reference but hereinafter ACV) via anoxidative cyclization step to isopenicillin N, epimerization topenicillin N and oxidative ring expansion to desacetoxycephalosporin C.##STR7## The activity of the racemase agent in a cell-free extract of C.acremonium was first recognized by Konomi et al, Biochem. J. Vol. 184, p427-430, 1979, and confirmed by Baldwin et al, Biochem J. Vol. 194,649-651, 1981, and Jayatilake et al, Biochem. J. Vol. 194, 649-647, 1981who also recognized the extreme lability of the racemase agent so thatrecovery of the racemase agent per se is believed to be impossible. Thelability of the racemase agent is believed to preclude use of cell-freeextracts of C. acremonium for high yield commercial production ofcephalosporins.

It is, therefore, an object of the present invention to provide anintegrated cell-free process for producing a cephalosporin compound froma peptide of the general formula ##STR8## where R₁ is hydrogen, a loweralkyl or functionalized carboxylic group, and R₂ is hydrogen or a loweralkyl group, using stable cell-free extracts from prokaryotic organisms.

These and other objects of the invention will be apparent from thefollowing description of the preferred embodiments.

SUMMARY OF THE INVENTION

It has now been discovered that certain cell-free extracts ofprokaryotic organisms such as Streptomyces clavuligerus, Streptomycescattleya and Streptomyces lipmanii, can be separated into threefractions by a three stage treatment to provide three stable andseparate enzymes: (a) epimerase (MW approx. 60,000) which may be used,for example, to epimerize isopenicillin N to penicillin N; (b) cyclase(MW approx. 36,500) which may be used, for example, to cyclize ACV toisopenicillin N; and (c) ring expansion enzyme (MW approx. 29,000) whichmay be used, for example, to ring expand penicillin N todeacetoxycephalosporin C.

Thus, by one aspect of this invention there is provided a process forproducing unnatural cephalosporins of the formula ##STR9## where R₁ =H,lower alkyl, or functionalized carboxylic and R₂ =H or lower alkyl andderivatives thereof, comprising reacting a starting material comprisingL-α-aminoadipyl-L-cysteinyl-D-valine and analogs thereof in which anamino acid is substituted for the valine moiety, with cyclase, epimeraseand a ring expansion enzyme isolated from a cell-free extract of aprokaryotic organism for sufficient time and in the presence ofsufficient co-factors to produce said cephalosporins.

By another aspect of this invention there is provided a process forisolating cyclase, epimerase and a ring expansion enzyme from acell-free extract of a prokaryotic organism comprising:

(a) precipitating contaminating proteins from said cell-free extract byaddition of ammonium sulfate to 40% saturation;

(b) separating precipitated protein from a supernatant;

(c) adding further ammonium sulfate to 70% saturation to saidsupernatant thereby precipitating desired said enzymes;

(d) suspending said precipitated enzymes in pH 7 buffer; and

(e) chromatographically separating the desired enzymes from each other.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following description, reference will be made particularly to theconversion of ACV (1) to deacetoxycephalosporin which is useful as anantibiotic as such or as a starting compound for the production ofcephalosporin antibiotics such as Cephalexin®. It will be appreciated,however, that the biochemical techniques of the present invention areequally applicable to other starting materials and it is within thepurview of the present invention to substitute the valine moiety in thepreferred ACV starting material with any of the readily available aminoacids for conversion to the analogous cephalosporins which are useful asantibiotics, or as starting materials for antibiotics such as Cefoxitin®or Ceftizoxime®. Thus, the starting material may be regarded as havingthe general formula (4) ##STR10## where R₁ and R₂ are as hereinbeforedescribed. The amino acids which may be used thus include:

    ______________________________________                                        R.sub.1        R.sub.2   Compound                                             ______________________________________                                        H              CH.sub.3  Valine                                               H              H         α-aminobutyric acid                            H              C.sub.2 H.sub.5                                                                         allo isoleucine                                      CH.sub.3       CH.sub.3  isoleucine                                           COOH           H         glutamic acid                                         ##STR11##     H         arginine                                               CONH.sub.2   H         glutamine                                            CH.sub.2 CH.sub.2 NH.sub.2                                                                   H         lysine                                               ______________________________________                                    

The naturally-occurring beta-lactam compounds are formed as secondarymetabolites of both eukaryotic and prokaryotic organisms. Simply stated,a eukaryote is a higher life form, and it has a more complicated cellstructure, which restricts the types of compounds that can besynthesized or metabolized. Examples of eukaryotic beta-lactam-producingorganisms are the fungi Penicillium chrysogenum and Cephalosporiumacremonium. A prokaryote, on the other hand, is a lower, earlier, lifeform, with a more primitive cell structure, which allows a greatervariety of chemical transformations to take place. This suggests, againsimply, that prokaryotes are more versatile at organic synthesis thanare eukaryotes, provided that this versatility can be understood andcontrolled. Examples of prokaroytic beta-lactam-producing organisms arethe actinomycetes Streptomyces clavuligerus, S. cattleya and S.lipmanii.

As an illustration of the differing capabilities of eukaryotic andprokaryotic beta-lactam-producing organisms, P. chrysogenum, aeukaryote, synthesizes ACV and converts this peptide sequentially topenicillin as the only stable beta-lactam-containing end product. C.acremonium, also a eukaryote, synthesizes the same tripeptide andconverts this peptide sequentially to penicillin and cephalosporin. Incontrast, the prokaryote S. clavuligerus synthesizes penicillin,cephalosporin and cephamycin from one amino acid-containing precursorand, at the same time, clavulanic acid, from a completely differentprecursor. The prokaryote S. cattleya synthesizes penicillin andcephalosporin from one precursor and, at the same time, the carbapenemthienamycin, from a different precursor.

S. clavuligerus, for example, is a well known microorganism and severalstrains are available, on an unrestricted basis, from the NorthernRegional Research Laboratory, Peoria, Ill., U.S.A. under the name NRRL3585, among others. Other prokaryotic organisms, as described above, areequally freely available. The NRRL 3585 organism must be cultured in amedium and under conditions conducive to the production of β-lactamcompounds, as described in more detail hereinafter.

There are several methods for cell breakage prior to obtaining acell-free extract, including French pressure cell, Omnimixer-plasticbeads and the preferred sonication. The preferred treatment comprisessonication for 30 seconds on 48 hour washed cells, followed bycentrifugation. The supernatant from this treatment is designated "crudecell-free extract". The crude extract may be separated into three enzymefractions in a three stage treatment. In the first stage, contaminatingproteins are precipitated by addition of ammonium sulfate to 40%saturation, and separated from the supernatant by centrifugation orother conventional means. Addition of more ammonium sulfate to 70%saturation precipitates the desired enzyme activities. The resultingpellet, suspended in pH 7 buffer is termed "salt-precipitated cell-freeextract" (SPCFX). This SPCFX retains all the desired enzyme activities,and shows reduced baseline contamination in HPLC assays. In the secondstage, the epimerase (isopenicillin N→penicillin N) (MW 60,000) iscleanly separated from the cyclase (ACV→isopenicillin N) (MW 36500) andring expansion (penicillin N→desacetoxycephalosporin C) (MW 20,000)enzymes, by gel filtration chromatography of the SPCFX on, for example,Sephadex®G-200 (Pharmacia, Sweden). In the third stage, the cyclase andring expansion enzymes are separated by ion exchange chromatography on,for example, DEAE Trisacryl resin (sold by L.K.B., Sweden). A 100-foldpurification of the cyclase is achieved in this manner. Thus, for thefirst time three distinct enzyme reagents each having a differentenzymatic activity and physical characteristics (e.g., differentmolecular weights) and which are stable over an extended period of time(of the order of months) under suitable storage conditions oftemperature and pH (preferably about -20° C. and pH7) have beenprepared. The enzymes may be stored and used quite separately or may bestored and used as a mixture as required.

Analogous treatment using SPCFX from C. acremonium yields the cyclaseand ring expansion enzymes only. As noted above the epimerase isentirely absent due to its extreme lability.

Following preparation of the three enzymes, ACV dimer or an analogthereof as described above, may be reacted therewith under aerobicconditions, in the presence of the required co-factors such as ferrousions usually in the form of ferrous sulfate, an antioxidant such asascorbic acid, a reducing agent such as dithiothreitol (DTT) and aco-substrate such as α-ketoglutarate, for sufficient time at about 20°C. and at a suitable pH of about 7 to produce desacetoxycephalosporin Cor an analog thereof.

EXAMPLE 1 Production of SPCFX

(a) Culture of S. clavuligerus

Streptomyces clavuligerus NRRL 3585 was maintained on a sporulationmedium composed of tomato paste, 20 g; oatmeal, 20 g; agar, 25 g, in 1liter of distilled water, pH 6.5.

Inoculated plates were incubated 7-10 days at 28° C. Spores were scrapedoff into sterile distilled water (5 ml/plate) and used to inoculate, 2%v/v, 25 ml/125 ml flask, seed medium of the following composition:glycerol, 10 ml; sucrose, 20 g; soy flour, 15 g; yeast extract, 1 g;tryptone, 5 g; K₂ HPO₄, 0.2 g in 1 liter of distilled water, pH 6.5.Inoculated seed medium was incubated for 3 days and used to inoculate,2% v/v, 100 ml amounts of production medium in 500 ml flasks. Productionmedium consisted of soluble starch, 10 g; L asparagine, 2 g;3-N-morpholinopropane-sulfonic acid, 21 g; MgSO₄.7H₂ O, 0.6 g; K₂ HPO₄,4.4 g; FeSO₄.7H₂ O, 1 mg; MnCl₂.4H₂ O, 1 mg; ZnSO₄.7H₂ O, 1 mg; andCaCl₂.2H₂ O, 1.3 mg in 1 liter of H₂ O, pH 6.8. Inoculated productionmedium was incubated 40-48 h and the cells were then collected byfiltration and used to prepare cell-free extracts. All incubations wereat 27° C. on a gyrotory shaker (250 rpm, 19 mm eccentricity).

(b) Preparation of Cell-Free Extracts

Cell-free extracts were prepared by washing 40-48 h cells of S.clavuligerus in 0.05M Tris-HCl buffer, pH b 7.0+0.1 mM dithiothreitol(DTT) (100 ml/100 ml culture). Washed cells were resuspended to 1/10 ofthe original culture volume in the same buffer and disrupted bysonication in an ice water bath for 2×15 sec at maximum intensity (300watts, Biosonik III, Bronwill Scientific). Broken cell suspensions werecentrifuged 1 h at 100,000 xg. All cell-free extracts were stored frozenat -20° C.

Salt-precipitated cell-free extract was prepared by gradual addition ofstreptomycin sulfate to cell-free extract with gentle stirring at 4° C.to a final concentration of 1%, w/v. After 15 min at 4° C., precipitatednucleic acid was removed by centrifugation for 15 min at 15,000 xg.Solid ammonium sulfate was then gradually added to the supernatant withgentle stirring at 4° C. until 40% saturation was reached. After 15 minat 4° C. the suspension was centrifuged as above and the pelletdiscarded. Additional ammonium sulfate was then added to thesupernatant, as above, until 70% saturation was reached. Followingcentrifugation, the pellet was resuspended to its original volume in0.05M Tris-HCl buffer pH 7.0 containing 0.1 mM DTT. The enzyme solutionwas then concentrated to 1/10 of the original volume by ultrafiltrationwith an Amicon®PM-10 filter.

Cyclization Assay System

Cyclization activity of enzyme preparations was measured in reactionmixtures containing: bis-δ-(L-α-aminoadipyl-L-cysteinyl-D-valine) (ACV)₂0.306 l mM, DDT 4 mM, Na ascorbate 2.8 mM, FeSO₄ 45 μM, tris-HCl buffer0.05M, pH 7.0, enzyme preparation 0.03-0.3 ml, final volume 0.4 ml.Reaction mixtures were incubated at 20° C. for up to 4 hours and stoppedby cooling on ice or by the addition of 0.4 ml methanol.

Ring Expansion Assay System

Ring expansion activity was followed using the cyclization assay systemdescribed above but supplemented with ATP 0.5 mM, α-ketoglutarate 1 mM,KCl 7.5 mM, and MgSO₄ 7.5 mM. Total volume and incubation conditionswere the same as for the cyclization assay.

EXAMPLE 2 Separation of Enzyme Fractions

(a) Separation of Epimerase by Gel Filtration Chromatography of SPCFX

2.5 ml of SPCFX was applied to a Sephadex®G-200 superfine column (2.5cm×40 cm) which had been equilibrated in 0.05M Tris-HCl buffer pH 7.0containing 0.1 mM DTT. The column was eluted with the same buffer and2.5 ml fractions were collected. Fractions were monitored for protein bymeasuring UV absorption at 280 nm, and were assayed for cyclase,epimerase and ring expansion activities as described previously. Activefractions were pooled and concentrated by ultrafiltration using anAmicon®PM-10 filter.

(b) Separation of Cyclase and Ring Expansion Enzyme by Ion ExchangeChromatography of SPCFX

2.5 ml of SPCFX was applied to a DEAE-Trisacryl® column (1.6×25 cm)which had been equilibrated in 0.1M Tris-HCl buffer pH 7.0 containing0.1 mM DTT. The column was washed with 50 ml of the above buffer andthen eluted with a linear gradient of 150 ml each of initial startingbuffer vs 0.4M Tris-HCl buffer pH 7.0 containing 0.1 mM DTT. 2.5 mlfractions were collected and monitored for protein content by measuringUV-absorption at 280 nm. Fractions were also monitored for conductivityand were assayed for cyclization, epimerase and ring expansion activityas described previously. Active fractions were pooled and concentratedand desalted by ultrafiltration using an Amicon®PM-10 filter.

Both separations were performed at 4° C., and the enzyme products werestored at -20° C. or lower as they were found to lose activity overnightat room temperature.

EXAMPLE 3 Preparation of ACV and Related Compounds

N-BoC-S-trityl-L-cysteine was coupled with the benzhydryl ester ofD-valine to give a fully protected dipeptide 5. ##STR12## A 15 minutetreatment with anhydrous formic acid at room temperature led tocrystalline, partially protected peptide 6. ##STR13## Conversion tofully protected ACV (7) ##STR14## was achieved by coupling peptide (6)with (8) ##STR15## Deprotection of (7) was achieved in two stages:

(a) removal of the trityl group, with iodine in methanol;

(b) removal of all other protecting groups by overnight treatment withformic acid, leading to ACV disulfide (9). The ACV is best stored inthis form and may be readily converted to ACV (1), as needed, withdithiothreitol. This synthesis is readily adaptable to systematicmodification of the aminoadipyl moiety and compounds such asN-acetyl-ACV and its cyclic analog N-acetyl isopenicillin N, may besimilarly prepared from N-acetyl-L-α-aminoadipic acid alpha-benzhydrylester as the starting material.

EXAMPLE 4 Preparation ofδ-(L-α-aminoadipyl)-L-cysteinyl-D-alloisoleucine (ACI) ##STR16##

This compound was prepared from L-α-aminoadipic acid, L-cysteine andD-alloisoleucine, as described for the synthesis of the naturalcephalosporin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine by S.Wolfe and M. G. Jokinen, Canadian Journal of Chemistry, Volume 57, pages1388-1396, 1979. This led, successively, to the fully protectedtripeptide (R₁ =t-butoxycarbonyl, R₂ =benzhydryl, R₃ =trityl), m.p.91°-93° (ethyl acetate-petroleum ether), R_(f) 0.54 (methylenechloride-ethyl acetate, 9:1; yellow with palladium chloride), thedetritylated compound (R₁ =t-butoxycarbonyl, R₂ =benzhydryl, R₃=disulfide), m.p. 114°-116° (methanol), R_(f) 0.76 (methylenechloride-ethyl acetate, 4:1, yellow with palladium chloride), and thecompletely deprotected compound (R₁ =R₂ =H, R₃ =disulfide), R_(f) =0.22(methyl ethyl ketone-water-acetic acid, 4:1:1, purple with ninhydrin), ¹Hmr (D₂ O) δ: 0.90 (3H, d, 6 Hz), 0.91 (3H, 5, 7 Hz), 1.30 (2H, m), 1.73(2H, br t), 1.88 (2H, br t), 2.01 (1H, m), 2.39 (2H, br t), 3.00 (1H, q,8, 15 Hz), 3.16 (1H, q, 5, 15 Hz), 3.76 (1H, t, 6 Hz), 4.40 (1H, d, 4Hz), 4.73 (1H, br s). The latter compound is converted into the activeform (R₁ =R₂ =R₃ =H) upon treatment with dithiothreitol.

EXAMPLE 5 Preparation ofδ-(L-α-aminoadipyl)-L-cysteinyl-D-α-aminobutyrate (ACAb) ##STR17##

This compound was prepared, as in Example 4, via the intermediates R₁=t-butoxycarbonyl, R₂ =benzyhydryl, R₃ =trityl: R_(f) 0.63(toluene-ethyl acetate, 2:1); R₁ =t-butoxycarbonyl, R₂ =benzhydryl, R₃=disulfide: R_(f) 0.48 (toluene-ethyl acetate, 2:1); and R₁ =R₂ =H; R₃=disulfide: R_(f) =0.1 (methyl ethyl ketone-water-acetic acid, 4:1:1), ¹Hmr (D₂ O) δ: 0.91 (3H, t, 7.5 Hz), 1.59-2.00 (6H, m), 2.41 (2H, t, 7Hz), 3.97 (1H, q, 8.5, 14 Hz), 3.21 (1H, q, 5, 14 Hz), 3.75 (1H, t, 7Hz), 4.18 (1H, q, 5, 8.5 Hz), 4.73 (1H, m). This last compound isconverted into the active form (R₁ =R₂ =R₃ =H) upon treatment withdithiothreitol.

EXAMPLE 6 Cyclization of ACV

To 0.4 ml of reaction mixture were added 0.9 mM of ACV dimer as producedin Example 3, 50.0 mM Tris-HCl pH 7.0 buffer and a mixture of the threeenzymes as produced in Example 1 from a cell-free extract of S.clavuligerus, together with 45.0 μM ferrous sulfate and 2.8 mM ascorbicacid as optimized amounts of essential co-factors. DTT was added inexcess of the amount required to reduce ACV dimer to ACV monomer. Thereaction was continued for approximately 2 hours at 20° C. and thenterminated by addition of 0.4 ml methanol to precipitate protein. It wasfound, by bioassay and HPLC procedures (described in more detailhereinafter) that the peptide had been converted to a mixture ofisopenicillin N and penicillin N. Ring expansion to a cephalosporin didnot occur. The experiment was repeated with the addition of 1 mM of astandard oxygenase type enzyme co-factor, alpha-ketoglutarate, and inthis case it was found that the ACV was converted todesacetoxycephalosporin C.

EXAMPLE 7

The procedures of Example 6 were repeated using L-aspartyl, L-glutamyl,D-α-aminoadipyl, adipyl, glycyl-L-α-aminoadipyl andN-acetyl-L-α-aminoadipyl-containing peptides. It was found that theL-aspartyl, L-glutamyl and D-α-aminoadipyl containing peptides did notcyclize. Cyclization was observed with adipyl, glycyl-L-α-aminoadipyland N-acetyl-L-α-aminoadipyl containing peptides. The adipyl compoundgave ca 20% cyclization to the corresponding penicillin,carboxybutylpenicillin but SPCFX converted the glycyl and N-acetylcompounds to penicillin N and isopenicillin N, via an initialdeacylation of these peptides to ACV. Purified cyclase from S.clavuligerus did not cyclize the glycyl-L-α-aminoadipyl containingpeptide. These results suggest that the enzymatic conversion of an ACVanalog to an unnatural cephalosporin nucleus requires (i) aδ-L-α-aminoadipyl side chain and (ii) an enzyme system containing theepimerase. A prokaryotic system is, therefore, required. Modification ofthe valinyl moiety, as noted above, has been considered in detail.Substrates modified in the valinyl moiety such as: ##STR18## where R₁ isH, a lower alkyl or functionalized carboxylic group; and R₂ is H or alower alkyl group may be cyclized with carbon-sulfur bond formation withretention of configuration at the beta carbon of the valine analog,leading to isopenicillin N analogs of the type: ##STR19## Followingepimerization to penicillin N analogs of the type: ##STR20## ringexpansion leads to cephalosporin analogs of the type: ##STR21## withtransfer of the beta carbon atom attached to C₂ of (11) into C₂ of thesix membered ring.

EXAMPLE 8 Bioassay of Beta-lactam Compounds

Antibiotic in reaction mixtures was estimated by the agar diffusionmethod. Cyclization reaction mixtures were bioassayed using Micrococcusluteus ATCC 9341 and Escherichia coli Ess as indicator organisms. Ringexpansion reaction mixtures were bioassayed using E. coli Ess asindicator organism in agar plates supplemented with penicillinase at2×10⁵ units/ml.

High Performance Liquid Chromatography (HPLC)

Methanol inactivated reaction mixtures were centrifuged at 12,000 xg for5 min to remove precipitated protein before analysis. Thechromatographic equipment used was: M-6000A pump, UK-6 injector, M-480variable wavelength detector, M-420 data module and Bondapak-C18 column(Rad Pak A in a Z module) as stationary phase. All equipment was fromWaters Scientific Co., Mississauga, Ontario. The mobile phase consistedof methanol/0.05M potassium phosphate buffer, pH 4.0. The methanolcontent of the mobile phase depended upon the particular separation. Ashort precolumn (packed with Bondapak C₁₈ /Corasil) was used to guardthe main column. UV-absorbing material was detected at 220 nm at asensitivity of 0.02 AUFS.

EXAMPLE 9 Cyclization and Ring Expansion of Unnatural Peptide Substrates

The procedure of Example 6 was repeated with ACV analogs in which valinewas replaced by alpha-aminobutyric acid (R₁ =R₂ =H) and allo-isoleucine(R₁ =H, R₂ =C₂ H₅), as follows:

(AC-aminobutyrate)₂ (ACAB)₂ and (AC-isoleucine)₂ (ACI)₂ were dissolvedin water neutralized and lyophilized in 0.1 and 1.0 mg amounts. Thesepeptides were then used as substrates in cyclization and ring expansionassays as follows: One hundred micrograms of (ACV)₂ from Example 6 wasused as substrate in a cyclization and a ring expansion assay systemusing 0.1 ml of salt-precipitated cell-free extract as enzyme source ineach case. (Final concentration of (ACV)₂ is 0.306 mM). Identicalcyclization and ring expansion assays were set up in which 100 μg(ACAB)₂ or 1.0 mg (ACI)₂ replaced the (ACV)₂ as substrate and 0.3 ml ofsalt precipitated cell-free extract was used as enzyme source. Nosubstrate controls were also prepared. The reaction mixtures wereincubated for 2 h at 20° C. At the end of incubation 20 μl amounts ofthe cyclization reaction mixtures were bioassayed versus M. luteus andE. coli Ess; 20 μl amounts of the ring expansion reaction mixtures werebioassayed versus E. coli Ess plus and minus penicillinase.

The remaining reaction mixtures were then mixed with an equal volume ofmethanol and centrifuged in preparation for HPLC analysis.

Cyclization and ring expansion reaction mixtures containing (ACI)₂ assubstrate and also the no substrate controls were analyzed using amobile phase of 20% Methanol/80% KH₂ PO₄, 0.05M adjusted to pH 4.0 withH₃ PO₄. Twenty microliter amounts were injected and eluted at a flowrate of 2 ml/min.

Cyclization and ring expansion reaction mixtures containing (ACV)₂,(ACI)₂ and (ACAB)₂ as substrates and also the no substrate controls werethen analyzed using a mobile phase of 5% Methanol/95% KH₂ PO₄, 0.05Madjusted to pH 4.0 with H₃ PO₄. Twenty microliter amounts were injectedand eluted at a flow rate of 2 ml/min for 5 min rising to 3 ml/min by 7min and remaining at 3 ml/min for the rest of the analysis time.

RESULTS AND DISCUSSION

Results of biological assays of the reaction mixtures are seen inTable 1. Cyclization of (ACV)₂ results in formation of a bioactiveproduct. The zone size produced on E. coli Ess agar plates (28.0 mm) isequivalent to the zone size which a cephalosporin C solution at 29.3μg/ml would produce. Cyclization of (ACAB)₂ produces a bioactive productwith antibiotic activity equivalent to a 0.9 μg/ml solution ofcephalosporin C against E. coli Ess. Similarly cyclization of (ACI)₂produces a bioactive product with antibiotic activity equivalent to a4.85 μg/ml solution of cephalosporin C against E. coli Ess. Ringexpansion assays containing (ACV)₂ result in formation ofpenicillinase-insensitive antibiotic which produces a zone size on E.coli Ess+penicillinase plates (22 mm) equivalent to a 7.6 μg/ml solutionof cephalosporin C. Ring expansion assays containing (ACAB)₂ do not formpenicillinase-insensitive antibiotic nor do they form any antibioticaffecting E. coli Ess. Since antibiotic activity was seen in (ACAB)₂-containing cyclization assay systems, this implies one of twothings: 1. The additional components in a ring expansion reactionmixture inhibit cyclization of ACAB, or 2. Ring expansion assayscontaining (ACAB)₂ produce a cephalosporin which does not affect E. coliEss. Ring expansion assays containing (ACI)₂ formpenicillinase-insensitive antibiotic which produces a zone size on E.coli Ess+penicillinase plates (12.5 mm) eqivalent to a 0.9 μg/mlsolution of cephalosporin C.

HPLC analysis of cyclization reaction mixtures containing (ACI)₂ assubstrate was carried out with a mobile phase of 20% methanol/80% KH₂PO₄, 0.05M pH 4.0. When compared with the no substrate control, (ACI)₂containing reaction mixtures showed a new peak at 2.66 min. Analysis ofring expansion reaction mixtures under the same conditions did not showany new peak because the region around 2.66 min was obscured by UVabsorbing material (α-ketoglutarate), present in both the no substratecontrol and in the test.

When the mobile phase was changed to 5% Methanol/95% KH₂ PO₄, 0.05M pH4.0, cyclization reaction mixtures containing (ACI)₂ now showed the newpeak to be at 11.26 min.

Ring expansion reaction mixtures containing (ACI)₂ showed the new peakto be somewhat (˜50%) reduced in size with a smaller peak running justin front of the main peak. This is expected since cephalosporinstypically run close to, but just in front of, their correspondingpenicillin.

Cyclization reaction mixtures containing (ACAB)₂ as substrate showed anew peak in the region of 2.33 min. The corresponding ring expansionreaction mixtures also show their new peak at 2.3 min. Since ringexpansion reaction mixtures do no show bioactivity despite the presenceof this new peak, we conclude that the cephalosporin is being formed butis of lower antibiotic activity against E. coli Ess than thecorresponding penicillin. Analysis of (ACV)₂ containing reactionmixtures shows that the natural product formed in cyclization reactionmixtures, a mixture of isopenicillin N and penicillin N [(iso)penicillinN], elutes at a retention time of 5.23 min. Ring expansion results inconversion of some of the penicillin to desacetoxy cephalosporin C whichruns with a retention time of 4.76 min and does not separate from(iso)penicillin N under these conditions.

Based on these studies, it is concluded that salt precipitated cell-freeextract from S. clavuligerus, can cyclize (ACI)₂ and (ACAB)₂ to formpenicillins, in addition to being able to cyclize the natural substrate,(ACV)₂. The unnatural penicillins so formed have chromatographiccharacteristics distinct from (iso)penicillin N and there is no evidencefor production of (iso)penicillin N in reaction mixtures containingunnatural peptide substrates. The same enzyme preparation can cause ringexpansion of the penicillin formed from (ACI)₂, resulting in formationof a new cephalosporin.

                  TABLE 1                                                         ______________________________________                                                     Zone of Inhibition (mm)                                          Substrate and           E. coli  E. coli Ess                                  Assay Conditions                                                                             M. luteus                                                                              Ess      + penicillinase                              ______________________________________                                        (ACV).sub.2 cyclization                                                                      29.0     28.0                                                  (ACV).sub.2 ring expansion                                                                            28.5     22.0                                         (ACAB).sub.2 cyclization                                                                      8.0     12.5                                                  (ACAB).sub.2 ring expansion                                                                            8.0     0                                            (ACI).sub.2 cyclization                                                                      13.0     20.0                                                  (ACI).sub.2 ring expansion                                                                            20.0     12.5                                         no substrate cyclization                                                                     ±     ±     ±                                         no substrate ring expansion                                                                  ±     ±     ±                                         ______________________________________                                    

We claim:
 1. A process for isolating cyclase, epimerase and a ringexpansion enzyme from a cell-free extract of a prokaryotic beta-lactamproducing organism comprising:(a) precipitating contaminating proteinsfrom said cell-free extract by addition of ammonium sulfate to 40%saturation; (b) separating precipitated protein from a supernatant; (c)adding ammonium sulfate to 70% saturation to said supernatant therebyprecipitating desired enzymes; (d) suspending said precipitated desiredenzymes in pH 7 buffer; (e) separating the epimerase by gel filtrationchromatography; and (f) separating the cyclase and ring expansionenzymes from each other by ion exchange chromatography.
 2. A process asclaimed in claim 1 wherein said prokaryotic organism is selected fromthe group comprising S. clavuligerus, S. cattleya and S. lipmanii.
 3. Aprocess as claimed in claim 2 wherein said prokaryotic organism is S.clavuligerus.